The long-term goal of this project is to develop a complete understanding of the spatial and temporal events involved in the regulation of gene expression in living ES cells as they transit from the pluripotent to the differentiated state. An understanding of the nuclear choreography of essential genes regulating the earliest stages of ES cell differentiation is integral to understanding the mechanisms that help shift the ES cell transcriptional program to cell-type specific gene expression and will provide the basis of identifying how alterations in these events alter development. Here, a series of Aims are proposed that will address questions relating to the impact of gene positioning and dynamics on transcriptional regulation using both live cell imaging and molecular approaches. Studies will focus on two transcriptional paradigms: (1) genes that go from the on to the off state, and (2) genes that switch to monoallelic expression upon commitment to differentiation. The nuclear position of a series of 4 genes encoding the transcription factors Oct4, Nanog, Sox2, and Klf4 will be examined, as transcriptional silencing of these genes serves as a trigger for the initiation of the differentiation process. Changes in the nuclear position/associations of one or more of these genes during the initiation of differentiation will be identified and characterized and the relationship to the transcriptional output of the genes will be examined at the single cell level. Based on these findings a live cell imaging system will be developed to allow the direct study of the spatial and temporal dynamics of the identified gene movements in living cells over time. This system will provide the visualization of the gene and its mRNA transcripts and will allow one to determine if changes in gene positioning/associations is the cause or effect of changes in the transcriptional output of these genes. Together, these proposed live cell imaging experiments will define the nature of allele dynamics and interactions, and provide insight into the functional consequences of such dynamics/interactions. In the final Aim the same differentiation paradigm will be used to identify genes that become monoallelically expressed upon differentiation. The active and inactive alleles will be characterized and a live cell imaging system will be developed to study the establishment of monoallelic gene expression. Together, the proposed studies will provide important insights into the underlying principles of nuclear organization and gene expression that will be critical to fully understanding the signals that regulate normal developmental progression and how they may be disrupted during disease progression and/or treatments.